Polymorphic forms of 3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione

ABSTRACT

Polymorphic forms of 3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione are disclosed. Compositions comprising the polymorphic forms, methods of making the polymorphic forms and methods of their use are also disclosed.

This application claims the benefit of U.S. provisional application60/499,723, filed Sep. 4, 2003, the contents of which are incorporatedby reference herein their entirety.

1. FIELD OF THE INVENTION

This invention relates to polymorphic forms of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione, compositions comprising thepolymorphic forms, methods of making the polymorphic forms and methodsof their use for the treatment of diseases and conditions including, butnot limited to, inflammatory diseases, autoimmune diseases, and cancer.

2. BACKGROUND OF THE INVENTION

Many compounds can exist in different crystal forms, or polymorphs,which exhibit different physical, chemical, and spectroscopicproperties. For example, certain polymorphs of a compound may be morereadily soluble in particular solvents, may flow more readily, or maycompress more easily than others. See, e.g., P. DiMartino, et al., J.Thermal Anal., 48:447-458 (1997). In the case of drugs, certain solidforms may be more bioavailable than others, while others may be morestable under certain manufacturing, storage, and biological conditions.This is particularly important from a regulatory standpoint, since drugsare approved by agencies such as the U.S. Food and Drug Administrationonly if they meet exacting purity and characterization standards.Indeed, the regulatory approval of one polymorph of a compound, whichexhibits certain solubility and physico-chemical (includingspectroscopic) properties, typically does not imply the ready approvalof other polymorphs of that same compound.

Polymorphic forms of a compound are known in the pharmaceutical arts toaffect, for example, the solubility, stability, flowability,fractability, and compressibility of the compound, as well as the safetyand efficacy of drug products comprising it. See, e.g., Knapman, K.Modern Drug Discoveries, 2000, 53. Therefore, the discovery of newpolymorphs of a drug can provide a variety of advantages.

U.S. Pat. Nos. 5,635,517 and 6,281,230, both to Muller et al., disclose3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione, whichis useful in treating and preventing a wide range of diseases andconditions including, but not limited to, inflammatory diseases,autoimmune diseases, and cancer. New polymorphic forms of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione canfurther the development of formulations for the treatment of thesechronic illnesses, and may yield numerous formulation, manufacturing andtherapeutic benefits.

3. SUMMARY OF THE INVENTION

This invention encompasses polymorphs of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. In certain aspects, theinvention provides polymorphs of the compound identified herein as formsA, B, C, D, E, F, G, and H. The invention also encompasses mixtures ofthese forms. In further embodiments, this invention provides methods ofmaking, isolating and characterizing the polymorphs.

This invention also provides pharmaceutical compositions and single unitdosage forms comprising a polymorph of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. The invention furtherprovides methods for the treatment or prevention of a variety ofdiseases and disorders, which comprise administering to a patient inneed of such treatment or prevention a therapeutically effective amountof a polymorph of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione.

4. BRIEF DESCRIPTION OF THE DRAWINGS

Specific aspects of the invention can be understood with reference tothe attached figures:

FIG. 1 provides a representative X-ray powder diffraction (XRPD) patternof Form A;

FIG. 2 provides a representative IR spectrum of Form A;

FIG. 3 provides a representative Raman spectrum of Form A;

FIG. 4 provides a representative thermogravimetric analysis (TGA) curveand a representative differential scanning calorimeter (DSC) thermogramof Form A;

FIG. 5 provides a representative moisture sorption/desorption isothermof Form A;

FIG. 6 provides a representative XRPD pattern of Form B;

FIG. 7 provides a representative IR spectrum of Form B;

FIG. 8 provides a representative Raman spectrum of Form B;

FIG. 9 provides a representative TGA curve and a representative DSCthermogram of Form B;

FIG. 10 provides representative TG-IR results of Form B;

FIG. 11 provides a representative moisture sorption/desorption isothermof Form B;

FIG. 12 provides a representative XRPD pattern of Form C;

FIG. 13 provides a representative IR spectrum of Form C;

FIG. 14 provides a representative Raman spectrum of Form C;

FIG. 15 provides a representative TGA curve and a representative DSCthermogram of Form C;

FIG. 16 provides representative TG-IR results of Form C;

FIG. 17 provides a representative moisture sorption/desorption isothermof Form C;

FIG. 18 provides a representative XRPD pattern of Form D;

FIG. 19 provides a representative IR spectrum of Form D;

FIG. 20 provides a representative Raman spectrum of Form D;

FIG. 21 provides a representative TGA curve and a representative DSCthermogram of Form D;

FIG. 22 provides a representative moisture sorption/desorption isothermof Form D;

FIG. 23 provides a representative XRPD pattern of Form E;

FIG. 24 provides a representative TGA curve and a representative DSCthermogram of Form E;

FIG. 25 provides a representative moisture sorption/desorption isothermof Form E;

FIG. 26 provides a representative XRPD pattern for a sample of Form F;

FIG. 27 provides a representative thermogram of Form F;

FIG. 28 provides a representative XRPD pattern of Form G;

FIG. 29 provides a representative DSC thermogram for a sample of Form G;

FIG. 30 provides a representative XRPD pattern of Form H;

FIG. 31 provides a representative TGA curve and a representative DSCthermogram of Form H;

FIG. 32 provides a representative XRPD pattern of Form B;

FIG. 33 provides a representative XRPD pattern of Form B;

FIG. 34 provides a representative XRPD pattern of Form B;

FIG. 35 provides a representative XRPD pattern of Form E;

FIG. 36 provides a representative XRPD pattern of polymorph mixture;

FIG. 37 provides a representative TGA curve of Form B;

FIG. 38 provides a representative TGA curve of Form B;

FIG. 39 provides a representative TGA curve of Form B;

FIG. 40 provides a representative TGA curve of Form E;

FIG. 41 provides a representative TGA curve of polymorph mixture;

FIG. 42 provides a representative DSC thermogram of Form B;

FIG. 43 provides a representative DSC thermogram of Form B;

FIG. 44 provides a representative DSC thermogram of Form B;

FIG. 45 provides a representative DSC thermogram of Form E;

FIG. 46 provides a representative DSC thermogram of polymorph mixture;

FIG. 47 provides a UV-Vis scan of dissolution medium;

FIG. 48 provides a UV-Vis scan of 0.04 mg/ml of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione in dissolution medium;

FIG. 49 provides a UV-Vis scan of 0.008 mg/ml of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione in dissolution medium;

FIG. 50 provides a calibration curve for 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione;

FIG. 51 provides a solubility curve of Form A;

FIG. 52 provides a solubility curve of Form B;

FIG. 53 provides an intrinsic dissolution of Forms A, B and E; and

FIG. 54 provides an intrinsic dissolution of Forms A, B and E.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Definitions

As used herein and unless otherwise indicated, the terms “treat,”“treating” and “treatment” refer to the alleviation of a disease ordisorder and/or at least one of its attendant symptoms.

As used herein and unless otherwise indicated, the terms “prevent,”“preventing” and “prevention” refer to the inhibition of a symptom of adisease or disorder or the disease itself.

As used herein and unless otherwise indicated, the terms “polymorph” and“polymorphic form” refer to solid crystalline forms of a compound orcomplex. Different polymorphs of the same compound can exhibit differentphysical, chemical and/or spectroscopic properties. Different physicalproperties include, but are not limited to stability (e.g., to heat orlight), compressibility and density (important in formulation andproduct manufacturing), and dissolution rates (which can affectbioavailability). Differences in stability can result from changes inchemical reactivity (e.g., differential oxidation, such that a dosageform discolors more rapidly when comprised of one polymorph than whencomprised of another polymorph) or mechanical characteristics (e.g.,tablets crumble on storage as a kinetically favored polymorph convertsto thermodynamically more stable polymorph) or both (e.g., tablets ofone polymorph are more susceptible to breakdown at high humidity).Different physical properties of polymorphs can affect their processing.For example, one polymorph might be more likely to form solvates ormight be more difficult to filter or wash free of impurities thananother due to, for example, the shape or size distribution of particlesof it.

Polymorphs of a molecule can be obtained by a number of methods known inthe art. Such methods include, but are not limited to, meltrecrystallization, melt cooling, solvent recrystallization, desolvation,rapid evaporation, rapid cooling, slow cooling, vapor diffusion andsublimation. Polymorphs can be detected, identified, classified andcharacterized using well-known techniques such as, but not limited to,differential scanning calorimetry (DSC), thermogravimetry (TGA), X-raypowder diffractometry (XRPD), single crystal X-ray diffractometry,vibrational spectroscopy, solution calorimetry, solid state nuclearmagnetic resonance (NMR), infrared (IR) spectroscopy, Ramanspectroscopy, hot stage optical microscopy, scanning electron microscopy(SEM), electron crystallography and quantitative analysis, particle sizeanalysis (PSA), surface area analysis, solubility, and rate ofdissolution.

As used herein to refer to the spectra or data presented in graphicalform (e.g., XRPD, IR, Raman and NMR spectra), and unless otherwiseindicated, the term “peak” refers to a peak or other special featurethat one skilled in the art would recognize as not attributable tobackground noise. The term “significant peaks” refers to peaks at leastthe median size (e.g., height) of other peaks in the spectrum or data,or at least 1.5, 2, or 2.5 times the median size of other peaks in thespectrum or data.

As used herein and unless otherwise indicated, the term “substantiallypure” when used to describe a polymorph of a compound means a solid formof the compound that comprises that polymorph and is substantially freeof other polymorphs of the compound. A representative substantially purepolymorph comprises greater than about 80% by weight of one polymorphicform of the compound and less than about 20% by weight of otherpolymorphic forms of the compound, more preferably greater than about90% by weight of one polymorphic form of the compound and less thanabout 10% by weight of the other polymorphic forms of the compound, evenmore preferably greater than about 95% by weight of one polymorphic formof the compound and less than about 5% by weight of the otherpolymorphic forms of the compound, and most preferably greater thanabout 97% by weight of one polymorphic forms of the compound and lessthan about 3% by weight of the other polymorphic forms of the compound.

5.2 Polymorphic Forms

This invention is directed to polymorphic forms of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione, which has the structureshown below:

This compound can be prepared according to the methods described in U.S.Pat. Nos. 6,281,230 and 5,635,517, the entireties of which areincorporated herein by reference. For example, the compound can beprepared through catalytic hydrogenation of 3-(4-nitro-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. 3-(4-Nitro-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione can be obtained by allowing2,6-dioxopiperidin-3-ammonium chloride to react with methyl2-bromomethyl-4-nitrobenzoate in dimethylformamide in the presence oftriethylamine. The methyl 2-bromomethyl-4-nitrobenzoate in turn isobtained from the corresponding methyl ester of nitro-ortho-toluic acidby conventional bromination with N-bromosuccinimide under the influenceof light.

Polymorphs of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione can be obtained bytechniques known in the art, including solvent recrystallization,desolvation, vapor diffusion, rapid evaporation, slow evaporation, rapidcooling and slow cooling. Polymorphs can be made by dissolving a weighedquantity of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione in various solvents atelevated temperatures. The solutions of the compound can then befiltered and allowed to evaporate either in an open vial (for fast hotevaporation) or in a vial covered with aluminum foil containing pinholes(hot slow evaporation). Polymorphs can also be obtained from slurries.Polymorphs can be crystallized from solutions or slurries using severalmethods. For example, a solution created at an elevated temperature(e.g., 60° C.) can be filtered quickly then allowed to cool to roomtemperature. Once at room temperature, the sample that did notcrystallize can be moved to a refrigerator then filtered. Alternatively,the solutions can be crash cooled by dissolving the solid in a solventat an increased temperature (e.g., 45-65° C.) followed by cooling in adry ice/solvent bath.

One embodiment of the invention encompasses Form A of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione. Form Ais an unsolvated, crystalline material that can be obtained fromnon-aqueous solvent systems. Another embodiment of the inventionencompasses Form B of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. Form B is a hemihydrated,crystalline material that can be obtained from various solvent systems.Another embodiment of the invention encompasses Form C of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione. Form Cis a hemisolvated crystalline material that can be obtained fromsolvents such as, but not limited to, acetone. Another embodiment of theinvention encompasses Form D of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. Form D is a crystalline,solvated polymorph prepared from a mixture of acetonitrile and water.Another embodiment of the invention encompasses Form E of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione. Form Eis a dihydrated, crystalline material. Another embodiment of theinvention encompasses Form F of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. Form F is an unsolvated,crystalline material that can be obtained from the dehydration of FormE. Another embodiment of the invention encompasses Form G of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione. Form Gis an unsolvated, crystalline material that can be obtained fromslurrying forms B and E in a solvent such as, but not limited to,tetrahydrofuran (THF). Another embodiment of the invention encompassesForm H of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. Form H is a partiallyhydrated crystalline material that can be obtained by exposing Form E to0% relative humidity. Each of these forms is discussed in detail below.

Another embodiment of the invention encompasses a composition comprisingamorphous 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione and crystalline3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione of formA, B, C, D, E, F, G or H. Specific compositions can comprise greaterthan about 50, 75, 90 or 95 weight percent crystalline3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione.

Another embodiment of the invention encompasses a composition comprisingat least two crystalline forms of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione (e.g., a mixture ofpolymorph forms B and E).

5.2.1 Form A

The data described herein for Form A, as well as for Forms B-H, wereobtained using the experimental methods described in Examples 6.3-6.7,provided below.

Form A can be obtained from various solvents, including, but not limitedto 1-butanol, butyl acetate, ethanol, ethyl acetate, methanol, methylethyl ketone, and THF. FIG. 1 shows a representative XRPD pattern ofForm A. The pattern is characterized by peaks, preferably significantpeaks, at approximately 8, 14.5, 16, 17.5, 20.5, 24, and 26 degrees 2θ.Representative IR and Raman spectra data are provided in FIGS. 2 and 3.

Representative thermal characteristics of Form A are shown in FIG. 4.TGA data show a small weight increase up to about 150° C., indicating anunsolvated material. Weight loss above 150° C. is attributed todecomposition. The DSC curve of Form A exhibits an endotherm at about270° C.

Representative moisture sorption and desorption data are plotted in FIG.5. Form A does not exhibit a significant weight gain from 5 to 95%relative humidity. Equilibrium can be obtained at each relative humiditystep. As the form dries from 95% back down to 5% relative humidity, ittends to maintain its weight such that at 5% relative humidity it hastypically lost only about 0.003% by weight from start to finish. Form Ais capable of remaining a crystalline solid for about 11 days whenstored at about 22, 45, 58, and 84% relative humidity.

Interconversion studies show that Form A can convert to Form B inaqueous solvent systems and can convert to Form C in acetone solventsystems. Form A tends to be stable in anhydrous solvent systems. Inwater systems and in the presence of Form E, Form A tends to convert toForm E.

When stored for a period of about 85 days under two differenttemperature/relative humidity stress conditions (room temperature/0%relative humidity (RH) and 40° C./93% RH), Form A typically does notconvert to a different form.

In sum, Form A is a crystalline, unsolvated solid that melts atapproximately 270° C. Form A is weakly or not hygroscopic and appears tobe the most thermodynamically stable anhydrous polymorph of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dionediscovered thus far.

5.2.2 Form B

Form B can be obtained from many solvents, including, but not limitedto, hexane, toluene, and water. FIG. 6 shows a representative XRPDpattern of Form B, characterized by peaks at approximately 16, 18, 22and 27 degrees 2θ.

Solution proton NMR confirm that Form B is a form of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione.Representative IR and Raman spectra are shown in FIGS. 7 and 8,respectively. Compared to Form A, the IR spectrum for Form B has peaksat approximately 3513 and 1960 cm⁻¹.

Representative DSC and TGA data for Form B are shown in FIG. 9. The DSCcurve exhibits endotherms at about 146 and 268° C. These events areidentified as dehydration and melting by hot stage microscopyexperiments. Form B typically loses about 3.1% volatiles up to about175° C. (per approximately 0.46 moles of water). Comparison of the IRspectrum of the volatiles with that of water indicates that they arewater (See FIG. 10). Calculations from TGA data indicate that Form B isa hemihydrate. Karl Fischer water analysis also supports thisconclusion.

Representative moisture sorption and desorption data are shown in FIG.11. Form B typically does not exhibit a significant weight gain from 5%to 95% relative humidity, when equilibrium is obtained at each relativehumidity step. As Form B dries from 95% back down to 5% relativehumidity, it tends to maintain its weight such that at 5% relativehumidity it typically has gained only about 0.022% by weight (about0.003 mg) from start to finish. Form B does not convert to a differentform upon exposure to about 84% relative humidity for about ten days.

Interconversion studies show that Form B typically converts to Form A ina THF solvent system, and typically converts to Form C in an acetonesolvent system. In aqueous solvent systems such as pure water and 10%water solutions, Form B is the most stable of the polymorphic forms of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione.However, it can convert to Form E in the presence of water. Desolvationexperiments show that upon heating at about 175° C. for about fiveminutes, Form B typically converts to Form A.

When stored for a period of about 85 days under two differenttemperature/relative humidity stress conditions (room temperature/0% RHand 40° C./93% RH), Form B does not convert to a different form.

In sum, Form B is a hemihydrated, crystalline solid that melts at about267° C. Interconversion studies show that Form B converts to Form E inaqueous solvent systems, and converts to other forms in acetone andother anhydrous systems.

5.2.3 Form C

Form C can be obtained from evaporations, slurries and slow cools inacetone solvent systems. A representative XRPD pattern of this form isshown in FIG. 12. The data are characterized by peaks at approximately15.5 and 25 degrees 2θ.

Solution proton NMR indicates that the 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione molecule is intact.Representative IR and Raman spectra are shown in FIGS. 13 and 14,respectively. The IR spectrum of Form C is characterized by peaks atapproximately 3466, 3373, and 3318 cm⁻¹. The Raman spectrum of Form C ischaracterized by peaks at about 3366, 3321, 1101, and 595 cm⁻¹.

Representative thermal characteristics for Form C are plotted in FIG.15. Form C loses about 10.02% volatiles up to about 175° C., indicatingit is a solvated material. Weight loss above about 175° C. is attributedto decomposition. Identification of volatiles in Form C can beaccomplished with TG-IR experiments. The representative IR spectrumcaptured after several minutes of heating, as depicted in FIG. 13, whencompared with a spectral library, shows acetone to be the best match.Calculations from TGA data show that Form C is a hemisolvate(approximately 0.497 moles of acetone). The DSC curve for Form C, shownin FIG. 15, exhibits endotherms at about 150 and about 269° C. Theendotherm at about 150° C. is attributed to solvent loss based onobservations made during hot stage microscopy experiments. The endothermat about 269° C. is attributed to the melt based on hot stageexperiments.

Representative moisture sorption and desorption balance data are shownin FIG. 17. Form C does not exhibit a significant weight gain from 5 to85% relative humidity, when equilibrium is obtained at each relativehumidity step up to 85% relative humidity. At 95% relative humidity,Form C experiences a significant weight loss of about 6.03%. As thesample dries from 95% back down to 5% relative humidity, the samplemaintains the weight achieved at the end of the adsorption phase at eachstep down to 5% relative humidity. Form C is capable of converting toForm B when stored at about 84% relative humidity for approximately tendays.

Interconversion studies show that Form C typically converts to Form A ina THF solvent system and typically converts to Form E in an aqueoussolvent system. In an acetone solvent system, Form C is the most stableform of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. Desolvation experimentsperformed on Form C show that upon heating at about 150° C. for aboutfive minutes, Form C will typically convert to Form A.

In sum, Form C is a crystalline, hemisolvated solid, which melts atapproximately 269° C. Form C is not hygroscopic below about 85% RH, butcan convert to Form B at higher relative humidities.

5.2.4 Form D

Form D can be obtained from evaporation in acetonitrile solvent systems.A representative XRPD pattern of the form is shown in FIG. 18. Thepattern is characterized by peaks at approximately 27 and 28 degrees 2θ.

Solution proton NMR indicates that the 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione molecule is intact.Representative IR and Raman spectra are shown in FIGS. 19 and 20,respectively. The IR spectrum of Form D is characterized by peaks atapproximately 3509, 2299, and 2256 cm⁻¹. The Raman spectrum of Form D ischaracterized by peaks at approximately 2943, 2889, 2297, 2260, 1646,and 1150 cm⁻¹.

Representative thermal characteristics for Form D are plotted in FIG.21. Form D loses about 6.75% volatiles up to about 175° C., indicating asolvated material. Weight loss above about 175° C. is attributed todecomposition. TG-IR experiments indicate that the volatiles are waterand acetonitrile. Calculations from TG data show that about one mole ofwater is present in the sample. A representative DSC curve for Form Dexhibits endotherms at about 122 and about 270° C. The endotherm atabout 122° C. is attributed to loss of volatiles based on observationsmade during hot stage microscopy experiments. The endotherm at about270° C. is attributed to the melt based on hot stage experiments.

Representative moisture sorption and desorption data are plotted in FIG.22. Form D does not exhibit a significant weight gain from 5 to 95%relative humidity when equilibrium is obtained at each relative humiditystep. As the form dries from 95% back down to 5% relative humidity, itmaintains its weight such that at 5% relative humidity the form hastypically gained only about 0.39% by weight (about 0.012 mg) from startto finish. Form A is capable of converting to Form B when stored atabout 84% relative humidity for approximately ten days.

Interconversion studies show that Form D is capable of converting toForm A in a THF solvent system, to Form E in an aqueous solvent system,and to Form C in an acetone solvent system. Desolvation experimentsperformed on Form D show that upon heating at about 150° C. for aboutfive minutes Form D will typically convert to Form A.

In sum, Form D is a crystalline solid, solvated with both water andacetonitrile, which melts at approximately 270° C. Form D is eitherweakly or not hygroscopic, but will typically convert to Form B whenstressed at higher relative humidities.

5.2.5 Form E

Form E can be obtained by slurrying 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione in water and by a slowevaporation of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione in a solvent system with aratio of about 9:1 acetone:water. A representative XRPD pattern is shownin FIG. 23. The data are characterized by peaks at approximately 20,24.5 and 29 degrees 2θ.

Representative thermal characteristics of Form E are plotted in FIG. 24.Form E typically loses about 10.58% volatiles up to about 125° C.,indicating that it is a solvated material. A second weight loss of anadditional about 1.38% was observed between about 125° C. and about 175°C. Weight loss above about 175° C. is attributed to decomposition. KarlFischer and TG-IR experiments support the conclusion that the volatileweight loss in Form E is due to water. The representative DSC curve forForm E exhibits endotherms at about 99, 161 and 269° C. Based onobservations made during hot stage microscopy experiments, theendotherms at about 99 and about 122° C. are attributed to loss ofvolatiles. The endotherm at about 269° C. is attributed to the meltbased on hot stage experiments.

Representative moisture sorption and desorption data are plotted in FIG.25. Form E typically does not exhibit a significant weight change from 5to 95% relative humidity when equilibrium is obtained at each relativehumidity step. As the sample dried from 95% back down to 5% relativehumidity, the sample continues to maintain weight such that at 5%relative humidity the sample has lost only about 0.0528% by weight fromstart to finish.

Interconversion studies show that Form E can convert to Form C in anacetone solvent system and to Form G in a THF solvent system. In aqueoussolvent systems, Form E appears to be the most stable form. Desolvationexperiments performed on Form E show that upon heating at about 125° C.for about five minutes, Form E can convert to Form B. Upon heating at175° C. for about five minutes, Form B can convert to Form F.

When stored for a period of 85 days under two differenttemperature/relative humidity stress conditions (room temperature/0% RHand 40° C./93% RH) Form E typically does not convert to a differentform. When stored for seven days at room temperature/0% RH, Form E canconvert to a new form, Form H.

5.2.6 Form F

Form F can be obtained by complete dehydration of Form E. Arepresentative XRPD pattern of Form F, shown in FIG. 26, ischaracterized by peaks at approximately 19, 19.5 and 25 degrees 2θ.

Representative thermal characteristics of Form A are shown in FIG. 27.The representative DSC curve for Form F exhibits an endotherm at about269° C. preceded directly by two smaller endotherms indicative of acrystallized form of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. The DSC thermogram does notshow any thermal events prior to the melt, suggesting that it is anunsolvated material.

5.2.7 Form G

Form G can be obtained by slurrying forms B and E in THF. Arepresentative XRPD pattern of this form, shown in FIG. 28, ischaracterized by a peak at approximately 23 degrees 2θ. Two other peaksunique to Form G appear at approximately 21 and 24.5 degrees 2θ.

Representative thermal characteristics of Form G are plotted in FIG. 29.A representative DSC curve for Form G exhibits an endotherm at about248° C. followed by a small, broad exotherm at about 267° C. No thermalevents are seen in the DSC thermogram at lower temperatures, suggestingthat it is an unsolvated material.

5.2.8 Form H

Form H can be obtained by storing Form E at room temperature and 0% RHfor about 7 days. A representative XRPD pattern is shown in FIG. 30. Thepattern is characterized by a peak at 15 degrees 2θ, and two other peaksat 26 and 31 degrees 2θ.

Representative thermal characteristics are shown in FIG. 31. Form Hloses about 1.67% volatiles up to about 150° C. Weight loss above about150° C. is attributed to decomposition. Karl Fischer data shows thatForm H typically contains about 1.77% water (about 0.26 moles),suggesting that the weight loss seen in the TG is due to dehydration.The DSC thermogram shows a broad endotherm between about 50° C. andabout 125° C., corresponding to the dehydration of Form H and a sharpendotherm at about 269° C., which is likely due to a melt.

When slurried in water with either Forms A or B, after about 14 daysForm H can convert to Form E. When slurried in THF, Form H can convertto Form A. When slurried in acetone, Form H can convert to Form C.

In sum, Form H is a crystalline solid, hydrated with about 0.25 moles ofwater, which melts at approximately 269° C.

5.3 Methods of Use and Pharmaceutical Compositions

Polymorphs of the invention exhibit physical characteristics that arebeneficial for drug manufacture, storage or use. All polymorphs of theinvention have utility as pharmaceutically active ingredients orintermediates thereof.

This invention encompasses methods of treating and preventing a widevariety of diseases and conditions using polymorphs of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Ineach of the methods, a therapeutically or prophylactically effectiveamount of the compound is administered to a patient in need of suchtreatment or prevention. Examples of such disease and conditionsinclude, but are not limited to, diseases associated with undesiredangiogenesis, cancer (e.g., solid and blood borne tumors), inflammatorydiseases, autoimmune diseases, and immune diseases. Examples of cancersand pre-cancerous conditions include those described in U.S. Pat. Nos.6,281,230 and 5,635,517 to Muller et al. and in various U.S. patentapplications to Zeldis, including application Ser. No. 10/411,649, filedApr. 11, 2003 (Treatment of Myelodisplastic Syndrome); Ser. No.10/438,213 filed May 15, 2003 (Treatment of Various Types of Cancer);Ser. No. 10/411,656, filed Apr. 11, 2003 (Treatment ofMyeloproliferative Diseases). Examples of other diseases and disordersthat can be treated or prevented using compositions of the invention aredescribed in U.S. Pat. Nos. 6,235,756 and 6,114,335 to D'Amato and inother U.S. patent applications to Zeldis, including Ser. No. 10/693,794,filed Oct. 23, 2003 (Treatment of Pain Syndrome) and Ser. No.10/699,154, filed Oct. 30, 2003 (Treatment of Macular Degeneration). Theentirety of each of the patents and patent applications cited herein isincorporated herein by reference.

Depending on the disease to be treated and the subject's condition,polymorphs of the invention can be administered by oral, parenteral(e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternalinjection or infusion, subcutaneous injection, or implantation),inhalation spray, nasal, vaginal, rectal, sublingual, or topical routesof administration and may be formulated, alone or together, in suitabledosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles appropriatefor each route of administration. Because individual polymorphs havedifferent dissolution, stability, and other properties, the optimalpolymorph used in methods of treatment may depend on the route ofadministration. For example, forms that are readily soluble in aqueoussolutions are preferably used to provide liquid dosage forms, whereasforms that exhibit great thermal stability may be preferred in themanufacture of solid dosage forms (e.g., tablets and capsules).

Although the physical characteristics of polymorphs can, in some cases,affect their bioavailability, amounts of the polymorphs that aretherapeutically or prophylactically effective in the treatment ofvarious disease and conditions can be readily determined by those ofordinary skill in the pharmacy or medical arts. In certain embodimentsof the invention, a polymorph is administered orally and in a single ordivided daily doses in an amount of from about 0.10 to about 150 mg/day,or from about 5 to about 25 mg/day. In other embodiments, a polymorph isadministered every other day in an amount of from about 0.10 to about150 mg/day, or from about 5 to about 25 mg/day.

The invention encompasses pharmaceutical compositions and single unitdosage forms that can be used in methods of treatment and prevention,which comprise one or more polymorphs of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione andoptionally one or more excipients or diluents. Specific compositions anddosage forms are disclosed in the various patents and patentapplications incorporated herein by reference. In one embodiment, asingle dosage form comprises a polymorph (e.g., Form B) in an amount ofabout 5, 10, 25 or 50 mg.

6. EXAMPLES

6.1 Polymorph Screen

A polymorph screen to generate the different solid forms of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione wascarried out as follows.

A weighed sample of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione (usually about 10 mg) wastreated with aliquots of the test solvent. Solvents were either reagentor HPLC grade. The aliquots were usually about 200 μL. Betweenadditions, the mixture was usually shaken or sonicated. When the solidsdissolved, as judged by visual inspection, estimated solubilities werecalculated. Solubilities were estimated from these experiments based onthe total solvent used to provide a solution. Actual solubilities mayhave been greater than those calculated due to the use of too-largesolvent aliquots or to a slow rate of dissolution.

Samples were created by generating solutions (usually about 30 mg in 20mL) at elevated temperatures, filtering, and allowing the solution toevaporate whether in an open vial (hot fast evaporation) or in a vialcovered with aluminum foil containing pinholes (hot slow evaporation).

Slurry experiments were also performed. Usually about 25 mg of solid wasplaced in either 3 or 5 mL of solvent. The samples were then placed onorbital shakers at either ambient temperature or 40° C. for 4-10 days.

Crystallizations were performed using various cooling methods. Solid wasdissolved in a solvent at an elevated temperature (e.g., about 60° C.),filtered quickly and allowed to cool to room temperature. Once at roomtemperature, samples that did not crystallize were moved to arefrigerator. Solids were removed by filtration or decantation andallowed to dry in the air. Crash cools were performed by dissolvingsolid in a solvent at an increased temperature (e.g., about 45-65° C.)followed by cooling in a dry ice/acetone bath.

Hygroscopicity studies were performed by placing portions of eachpolymorph in an 84% relative humidity chamber for approximately oneweek.

Desolvation studies were carried out by heating each polymorph in a 70°C. oven for approximately one week.

Interconversion experiments were carried out by making slurriescontaining two forms in a saturated solvent. The slurries were agitatedfor approximately 7-20 days at ambient temperature. The insoluble solidswere recovered by filtration and analyzed using XRPD.

6.2 Preparation of Polymorphic Forms

Eight solid forms of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione were prepared as describedbelow.

Form A was obtained by crystallization from various non-aqueous solventsincluding 1-butanol, butyl acetate, ethanol, ethyl acetate, methanol,methyl ethyl ketone, and tetrahydrofuran. Form B was also obtained bycrystallization from the solvents hexane, toluene and water. Form C wasobtained from evaporations, slurries, and slow cools in acetone solventsystems. Form D was obtained from evaporations in acetonitrile solventsystems. Form E was obtained most readily by slurrying3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione inwater. Form F was obtained by complete desolvation of Form E. It isfound to be an unsolvated, crystalline material that melts at about 269°C. Form G was obtained by slurrying forms B and E in THF. Form H wasobtained by stressing Form E at room temperature and 0% RH for 7 days.

6.2.1 Synthesis of Polymorphs B and E

Form B is the desired polymorph for the active pharmaceutical ingredient(API) of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. This form has been used inthe formulation of API into drug product for clinical studies. Threebatches were produced as apparent mixtures of polymorphs in thenon-micronized API of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione.

Development work was carried out to define a process that would generatepolymorph B from this mixture of polymorphs and could be implemented forstrict polymorphic controls in the validation batches and futuremanufacturing of API of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. Characterization ofpolymorphic forms produced during the work was performed by XRPD, DSC,TGA and KF.

A process was also developed for the large-scale preparation of Form E.Polymorph E material was prepared in order to carry out a comparisonwith polymorph B drug product in capsule dissolution testing of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione. 150 gof a mixture of polymorphs in 3 L of water was stirred at roomtemperature for 48 hours. The product was collected by filtration anddried at 25° C. for 24 hours under vacuum. XRPD, DSC, TGA, KF and HPLCanalyses confirmed that the material isolated was polymorph E.

In a preliminary work, it was demonstrated that stirring a suspension ofa mixture of polymorphs of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione with water at hightemperature (75° C.) for an extended period of time converted thismixture of polymorphs exclusively to form B. Several specific parameterswere identified including temperature, solvent volume and dryingparameters (temperature and vacuum). XRPD, DSC, TGA, KF and HPLCanalyses were used to characterize all of the batches. After completingthe optimization work, the optimized process was scaled-up to 100-200 gon three lots of API. Drying studies were carried out at 20° C., 30° C.and 40° C., and 65° C. with a vacuum of 150 mm of Hg. The results areshown in Tables 1-5.

The cooling and holding periods of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione slurry were studied. Theexperimental laboratory data suggests that polymorph B seems to beforming first, and overtime equilibration to polymorph E at RTconditions occurs, therefore generating a mixture of polymorphs B and E.This result supports the fact that polymorph B seems to be a kineticproduct, and that prolonged processing time converts the material topolymorph E resulting in a mixture of polymorphs B and E.

A laboratory procedure was developed to exclusively produce polymorph Bof 3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione. Theprocedure includes a stirred 10 volume water slurry at ˜75° C. for 6-24hours. The following preferred process parameters have been identified:

-   -   1. Hot slurry temperature of 70-75° C.    -   2. Product filtration of 3-(4-amino-1-oxo-1,3        dihydro-isoindol-2-yl)-piperidine-2,6-dione at 65-75° C.    -   3. Drying under vacuum at 60-70° C. is preferred for an        efficient removal of unbound water in 3-(4-amino-1-oxo-1,3        dihydro-isoindol-2-yl)-piperidine-2,6-dione wet cake.    -   4. The filtration step of 3-(4-amino-1-oxo-1,3        dihydro-isoindol-2-yl)-piperidine-2,6-dione may be a time        sensitive operation. The use of efficient solid-liquid        separation equipment is preferred.    -   5. Holding periods of water-wet cake of 3-(4-amino-1-oxo-1,3        dihydro-isoindol-2-yl)-piperidine-2,6-dione at KF higher than 5%        may cause the kinetic equilibrations of polymorph B to mixed        polymorphs of E and B.

Drying to KF<4.0% water was achieved in ˜3 hours (30-70° C., 152 mm Hg).Polymorphs B and E were distinguished by the water levels as measured byKF and TGA. The reference sample of polymorph B is micronized API. Inorder to make accurate comparison by XRPD samples were gently grindedbefore submission for analysis. This increases the clarity of theidentification of the polymorphic form. All samples were analyzed forXRPD, DSC, TGA, KF and HPLC.

TABLE 1 Preliminary Studies Reaction Results/ Amount conditions Analysisconclusion 2 g Water, rt, 48 h XRPD, DSC, Polymorph E TGA, KF 25 g Water, rt, 48 h XRPD, DSC, Polymorph E TGA, KF 5 g Water, 70-75° C.,XRPD, DSC, Polymorph B 24 h then rt 24 h TGA, KF 1 g 9:1 Acetone - XRPD,DSC, Polymorph water, Slow evpo. TGA, KF Mixture 1 g 175° C. 1 h in anXRPD, DSC, Polymorph A oven TGA, KF 0.5 g (poly- Water, rt, 24 h XRPD,DSC, Polymorph E morph A) TGA, KF 1 g poly- Water, rt, 48 h XRPD, DSC,Polymorph E morph B TGA, KF 1 g poly- Water, 70-75° C., XRPD, DSC,Polymorph B morph E 24 h TGA, KF 1 g Slurry in heptane XRPD, DSC, Nochange TGA, KF

TABLE 2 Optimization of Temperature, Time and Solvent Volume AmountWater Temp Time Results/ Amount (mL) (° C.) (h) conclusion 10 g 50 75 6Mix 10 g 50 75 24 Polymorph B 10 g 100 70 6 Polymorph B 10 g 100 70 14Polymorph B 10 g 100 70 21 Polymorph B 10 g 100 75 6 Polymorph B 10 g100 75 24 Polymorph B 10 g 100 75 6 Polymorph B 10 g 100 75 19 PolymorphB 10 g 100 75 14 Polymorph B 10 g 100 75 24 Polymorph B  5 g 100 75 18Polymorph B 10 g 100 80 6 Polymorph B 10 g 100 80 20 Polymorph B 10 g200 45 6 Polymorph B + E 10 g 200 45 24 Polymorph E 10 g 200 60 48Polymorph B 10 g 200 75 6 Mix 10 g 200 75 24 Polymorph B 10 g 200 75 13Polymorph B 10 g 200 75 24 Polymorph BOptimum conditions were determined to be 10 volumes of solvent (H₂O),70-80° C. for 6-24 hours.

TABLE 3 Holding Time Holding Holding Reaction Time Temp Results/ AmountConditions (h) (° C.) Conclusion  5 g Water, 70-75° C., 24 23-25Polymorph B 24 h 1 g Poly- Water, 70-75° C., 48 23-25 Polymorph E morphB 24 h  2 g Water, 40 mL 16 23-25 Polymorph E 150 g  Water, 3.0 L 2423-25 Polymorph E 150 g  Water, 3.0 L 48 23-25 Polymorph E 10 g Water,100 mL, 18 23-25 Polymorph B 24 h, 75° C. 10 g Water, 100 mL, 18 40Polymorph B 24 h, 75° C. 10 g Water, 200 mL, 14 −5 Mix 24 h, 75° C. 10 gWater, 200 mL, 14 23-25 Polymorph E 24 h, 75° C. 10 g Water, 200 mL, 1440 Mix 24 h, 75° C. 10 g Water, 100 mL, 21 23-25 Polymorph E 24 h, 75°C. 10 g Water, 100 mL, 21 40 Mix 24 h, 75° C. 10 g Water, 100 mL, 223-25 Mix 14 h, 75° C.Holding time gave mixed results and it was determined that the materialshould be filtered at 60-65° C. and the material washed with 0.5 volumeof warm (50-60° C.) water.

TABLE 4 Scale-up Experiments Amount Water Temp Time Results/ Amount (L)(° C.) (h) Conclusion 100 g 1.0 75 6 Polymorph B 100 g 1.0 75 22Polymorph B 100 g 1.0 75 6 Polymorph B 100 g 1.0 75 24 Polymorph B 100 g1.0 75 6 Polymorph B 100 g 1.0 75 22 Polymorph B

TABLE 5 Drying Studies Drying Drying Time Temp Vacuum KF§ Results/Amount (h) (° C.) (mm Hg) (%) Conclusion 100 g 0 — — 3.690 Polymorph B100 g 3 30 152 3.452 Polymorph B 100 g 8 30 152 3.599 Polymorph B 100 g0 — — 3.917 Polymorph B 100 g 5 40 152 3.482 Polymorph B 100 g 22 40 1523.516 Polymorph B 100 g 3 40 152 3.67 Polymorph B 100 g 22 40 152 3.55Polymorph B *Reaction Conditions: Water 1 L, 75° C., 22-24 h; §Averageof 2 runs.Drying studies determined that the material should be dried at 35-40°C., 125-152 mm Hg for 3 to 22 h or until the water content reaches ≦4%w/w.

For a large scale preparation of polymorph E (5222-152-B), a 5-L roundbottom flask was charged with 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione (150 g, 0.579 mol) and water(3000 mL, 20 vol). The mixture was mechanically stirred at roomtemperature (23-25° C.) for 48 h under nitrogen atmosphere.

Samples were taken after 24 h and 48 h before the mixture was filteredand air-dried on the filter for 1 h. The material was transferred to adrying tray and dried at room temperature (23-25° C.) for 24 h. KFanalysis on the dried material showed water content of 11.9%. Thematerial was submitted for XRPD, TGA, DSC and HPLC analysis. Analysisshowed the material was pure polymorph E.

For a large scale preparation of polymorph B (5274-104), a 2 L-3-neckedround bottom flask was charged with 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione (polymorph mixture, 100 g,0.386 mol) and water (1000 mL, 10.0 vol). The mixture was heated to 75°C. over approximately 30 minutes with mechanical stirring under nitrogenatmosphere.

Samples were taken after 6 h and 24 h before the mixture was allowed tocool to 60-65° C., filtered and the material washed with warm (50-60°C.) water (50 mL, 0.5 vol). The material was transferred to a dryingtray and dried at 30° C., 152 mm Hg for 8 h. KF analysis on the driedmaterial showed water content of 3.6%. After grinding the material wassubmitted for XRPD, TGA, DSC and HPLC analysis. Analysis showed thematerial was pure polymorph B. The results of the analyses are shown inFIGS. 32-46.

6.3 X-Ray Powder Diffraction Measurements

X-ray powder diffraction analyses were carried out on a ShimadzuXRD-6000 X-ray powder diffractometer using Cu Kα radiation. Theinstrument is equipped with a fine-focus X-ray tube. The tube voltageand amperage were set at 40 kB and 40 mA, respectively. The divergenceand scattering slits were set at 1° and the receiving slit was set at0.15 mm. Diffracted radiation was detected by a NaI scintillationdetector. A theta-two theta continuous scan at 3°/min (0.4 sec/0.02°step) from 2.5 degrees 2θ to 40 degrees 2θ was used. A silicon standardwas analyzed each day to check the instrument alignment.

X-ray powder diffraction analyses were also carried out using Cu Kαradiation on an Inel XRG-3000 diffractometer equipped with a curvedposition-sensitive detector. Data were collected in real time over atheta-two theta range of 120° at a resolution of 0.03°. The tube voltageand current were 40 kV and 30 mA, respectively. A silicon standard wasanalyzed each day to check for instrument alignment. Only the regionbetween 2.5 and 40 degrees 2θ is shown in the figures.

6.4 Thermal Analysis

TG analyses were carried out on a TA Instrument TGA 2050 or 2950. Thecalibration standards were nickel and alumel. Approximately 5 mg ofsample was placed on a pan, accurately weighed, and inserted into the TGfurnace. The samples were heated in nitrogen at a rate of 10° C./min, upto a final temperature of 300 or 350° C.

DSC data were obtained on a TA 2920 instrument. The calibration standardwas indium. Approximately 2-5 mg samples were placed into a DSC pan andthe weight accurately recorded. Crimped pans with one pinhole were usedfor analysis and the samples were heated under nitrogen at a rate of 10°C./min, up to a final temperature of 350° C.

Hot-stage microscopy was carried out using a Kofler hot stage mounted ona Leica Microscope. The instrument was calibrated using USP standards.

A TA Instruments TGA 2050 interfaced with a Nicolet model 560 Fouriertransform IR spectrophotometer, equipped with a globar source, XT/KBrbeamsplitter, and deuterated triglycine sulfate (DTGS) detector, wasutilized for TG-IR experiments. The IR spectrometer was wavelengthcalibrated with polystyrene on the day of use while the TG wastemperature and weight calibrated biweekly, using indium for thetemperature calibration. A sample of approximately 10 mg of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione wasweighed into an aluminum pan and heated from 25 to 30° C. to 200° C. ata rate of 20° C./min with a helium purge. IR spectra were obtained inseries, with each spectrum representing 32 co-added scans at aresolution of 4 cm⁻¹. Spectra were collected with a 17-second repeattime. TG/IR analysis data are presented as Gram-Schmidt plots and IRspectra linked to the time. Gram-Schmidt plots show total IR intensityvs. time; hence, the volatiles can be identified at each time point.They also show when the volatiles are detected. From the Gram-Schmidtplots, time points were selected and the IR spectra of these time pointsare presented in the stacked linked spectra. Each spectrum identifiesvolatiles evolving at that time point. Volatiles were identified from asearch of the HR Nicolet TGA vapor phase spectral library. The librarymatch results are also presented to show the identified vapor.

6.5 Spectroscopy Measurements

Raman spectra were acquired on a Nicloet model 750 Fourier transformRaman spectrometer utilizing an excitation wavelength of 1064 nm andapproximately 0.5 W of Nd:YAG laser power. The spectra represent 128 to256 co-added scans acquired at 4 cm⁻¹ resolution. The samples wereprepared for analysis by placing the material in a sample holder andpositioning this in the spectrometer. The spectrometer was wavelengthcalibrated using sulfur and cyclohexane at the time of use.

The mid-IR spectra were acquired on a Nicolet model 860 Fouriertransform IR spectrophotmeter equipped with a globar source XT/KBrbeamsplitter and a deuterated triglycine sulfate (DTGS) detector. ASpectra-Tech, Inc. diffuse reflectance accessory was utilized forsampling. Each spectrum represents 128 co-added scans at a spectralresolution of 4 cm⁻¹. A background data set was acquired with analignment mirror in place. A single beam sample data set was thenacquired. Subsequently, a log 1/R (where R=reflectance) spectrum wasacquired by rationing the two data sets against each other. Thespectrophotometer was calibrated (wavelength) with polystyrene at thetime of use.

6.6 Moisture Sorption/Desorption Measurements

Moisture sorption/desorption data were collected on a VTI SGA-100moisture balance system. For sorption isotherms, a sorption range of 5to 95% relative humidity (RH) and a desorption range of 95 to 5% RH in10% RH increments was used for analysis. The sample was not dried priorto analysis. Equilibrium criteria used for analysis were less than0.0100 weight percent change in 5 minutes with a maximum equilibrationtime of 3 hours if the weight criterion was not met. Data were notcorrected for the initial moisture content of the samples.

6.7 Solution Proton NMR Measurements

NMR spectra not previously reported were collected at SSCI, Inc, 3065Kent Avenue, West Lafayette, Ind. Solution phase ¹H NMR spectra wereacquired at ambient temperature on a Bruker model AM spectrometer. The¹H NMR spectrum represents 128 co-added transients collected with a 4μsec pulse and a relaxation delay time of 5 seconds. The free inductiondecay (FID) was exponentially multiplied with a 0.1 Hz Lorentzian linebroadening factor to improve the signal-to-noise ratio. The NMR spectrumwas processed utilizing GRAMS software, version 5.24. Samples weredissolved in dimethyl sulfoxide-d₆.

The scope of this invention can be understood with reference to theappended claims.

6.8 Intrinsic Dissolution and Solubility Studies

Intrinsic dissolution experiments were conducted on Form A (anhydrous),Form B (hemihydrate), and Form E (dihydrate) of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione. Equilibrium solubilityexperiments were conducted on Forms A and B. Aliquots were analyzed byultraviolet-visible spectrophotometry, and the solids remaining fromeach experiment were analyzed by X-ray powder diffraction (XRPD).

6.8.1 Experimental

6.8.1.1 Dissolution

Dissolution experiments were carried out in a VanKel VK6010-8dissolution apparatus equipped with a VK650A heater/circulator. Anintrinsic dissolution apparatus (Woods apparatus) was used. Samples werecompressed at 1.5 metric tons (1000 psi) for 1 min using the Woodsapparatus in a hydraulic press, giving a sample surface of 0.50 cm². Adissolution medium consisting of 900 mL HCl buffer, pH 1.8, with 1%sodium lauryl sulfate, was used for each experiment. The medium wasdegassed by vacuum filtration through a 0.22-μm nylon filter disk andmaintained at 37° C. The apparatus was rotated at 50 rpm for eachexperiment. Aliquots were filtered immediately using 0.2-μm nylonsyringe filters. In some cases, the undissolved solids were recoveredand analyzed by X-ray powder diffraction (XRPD).

6.8.1.2 Solubility

Equilibrium solubility experiments were conducted in a 100-mL,three-neck, round-bottom flask immersed in a constant temperature oilbath maintained at 25° C. A solid sample of 400-450 mg was stirred in 50mL of dissolution medium (HCl buffer, pH 1.8, with 1% sodium laurylsulfate) using a mechanical stir rod. Aliquots were filtered using0.2-μm nylon syringe filters and immediately diluted 1 mL→50 mL, then 5mL→25 mL with dissolution medium in Class A glassware, a final dilutionfactor of 250.

6.8.1.3 UV-Vis Spectrophotometry

Dissolution and solubility samples solutions were analyzed by a BeckmanDU 640 single-beam spectrophotometer. A 1.000-cm quartz cuvette and ananalysis wavelength of 228.40 nm were utilized. The detector was zeroedwith a cuvette filled with dissolution medium.

6.8.1.4 X-Ray Powder Diffraction

XRPD analyses were carried out on a Shimadzu XRD-6000 X-ray powderdiffractometer using Cu Kα radiation. The instrument is equipped with afine focus X-ray tube. The tube power and amperage were set at 40 kV and40 mA, respectively. The divergence and scattering slits were set at 1°and the receiving slit was set at 0.15 mm. Diffracted radiation wasdetected by a NaI scintillation detector. A theta-two theta continuousscan at 3°/min (0.4 sec/0.02° step) from 2.5 to 40° 2θ was used. Asilicon standard was analyzed each day to check the instrumentalignment. Samples were packed in an aluminum holder with siliconinsert.

6.8.2 Results

The results of these solubility and intrinsic studies are summarized inTable 6. Both the solubility and dissolution experiments were conductedin a medium of HCl buffer, pH 1.8, containing 1% sodium lauryl sulfate.Form A was found to be unstable in the medium, converting to Form E. Thesolubilities of Forms A, B, and E were estimated to be 6.2, 5.8, and 4.7mg/mL, respectively. The dissolution rates of Forms A, B, and E wereestimated to be 0.35, 0.34, and 0.23 mg/mL, respectively.

6.8.2.1 UV-Vis Spectrophotometry Method Development

A UV-Vis scan of the dissolution medium (blanked with an empty cuvette)was done to identify any interfering peaks. A small peak at 225 nm waspresent as shown in FIG. 47.

Solutions of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione at varying concentrationswere analyzed by UV-Vis spectrophotometry. A preliminary scan of a 1.0mg/mL solution was done, with the instrument blanked with dissolutionmedium. The solution was highly absorbing and noisy from 200-280 nm,making dilution necessary.

A 0.04 mg/mL solution of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione was then scanned from200-300 nm. The plot was still noisy between 200 and 230 nm as shown inFIG. 48. The sample was further diluted to 0.008 mg/mL. A wavelengthscan of 200-350 nm for this sample showed a peak a 228.4 nm with nointerference, as shown in FIG. 49. Therefore, a wavelength of 228.4 waschosen for analysis of the solubility and dissolution samples.

A six-point calibration curve was generated with standards of thefollowing concentrations: 0.001 mg/mL, 0.002 mg/mL, 0.005 mg/mL, 0.010mg/mL, 0.015 mg/mL, and 0.020 mg/mL (Notebook 569-90). A linearitycoefficient of R²=0.9999 was obtained as shown in FIG. 50.

6.8.2.2 Solubility

A sample consisting of 449.4 mg Form A was slurried in dissolutionmedium. Particle size was not controlled. Aliquots were taken at 7, 15,30, 60, 90, and 150 min. The concentration reached 6.0 mg/mL by thefirst time point. The highest concentration reached was 6.2 mg/mL, at 30min. From that point the concentration decreased, reaching 4.7 mg/mL at150 min as in FIG. 51. The solids remaining at the final time point wereanalyzed by XRPD and found to be Form E as shown in Table 7. No peaksattributed to Form A can be seen in the pattern. Since the concentrationdid not plateau at 4.7 mg/mL, the solubility of Form E may be lower thanthat.

A sample consisting of 401.4 mg Form B was slurried in dissolutionmedium. Particle size was not controlled. Aliquots were taken at 7, 15,30, 60, 90, 180, 420, and 650 min. Form B dissolved much more slowlythan Form A, reaching 3.3 mg/mL in 90 min. The concentration stabilizedat 5.6-5.7 mg/mL at the final three time points as in FIG. 52. Theremaining solids were shown to be Form B as in Table 7, suggesting FormB has good stability in water.

A summary of the solubilities is given in Table 6. The amounts dissolvedat each time point are shown in Tables 8 and 9.

TABLE 6 Summary of Results Average Intrinsic Intrinsic IntrinsicDissolution Form Solubility Dissolution #1 Dissolution #2 Rate Form A6.2 mg/mL 0.35 0.22^(a) 0.29^(a) Form B 5.8 mg/mL 0.35 0.32 0.34 Form E4.7 mg/mL 0.21 0.25 0.23 ^(a)The Form A dissolution experiment #2 mayhave converted to Form E on the surface of the disk, skewing the averagerate lower.

TABLE 7 Experimental Details Experiment Final Form Pressed Form A APressed Form B B Form A Solubility E Form B Solubility B Form ADissolution — Form A Dissolution A Form B Dissolution — Form BDissolution B Form E Dissolution E Form E Dissolution —

TABLE 8 Form A Solubility Time Point (min) Concentration (mg/mL) 7 6.0015 6.11 30 6.16 60 6.10 90 5.46 150 4.73

TABLE 9 Form B Solubility Time Point (min) Concentration (mg/mL) 7 1.6315 2.14 30 2.33 60 2.94 90 3.34 180 5.67 420 5.76 650 5.61

6.8.2.3 Intrinsic Dissolution

Approximately 200 mg each of Forms A and B were compressed into disks inthe Woods apparatus using 2 metric tons of pressure. The samples weresubsequently scraped out, ground gently, and analyzed by XRPD. The studyshowed that compression and grinding does not cause a form change ineither case. (See Table 7).

Two preliminary dissolution runs were performed. The disks fractured tosome extent in both experiments, compromising the requirement ofconstant surface area.

The first experiment of intrinsic dissolution that strictly followed theUSP chapter on intrinsic dissolution utilized approximately 150 mg eachof Forms A and B. Seven aliquots, beginning at 5 min and ending at 90min, were taken to maintain sink conditions. The experiment resulted inlinear dissolution profiles, with a rate of 0.35 mg per cm² per minutefor both forms. The Form E experiment was done later under the sameconditions and added to the graph for comparison. (See FIG. 53). TheForm E dissolution rate was 0.21 mg per cm² per minute, significantlylower than the dissolution rate of Forms A and B. This is in line withexpectations based on the solubility data. The crystal form of theremaining solids did not change in any case.

The second experiment utilized approximately 250 mg each of Forms A andB. The Form E experiment (135 mg) was done later and added to the graphfor comparison. (See FIG. 54). Nine aliquots were taken, beginning at 5min and ending at 150 min. The dissolution rates were 0 22, 0.32, and0.25 mg per cm² per minute, respectively, for Forms A, B, and E. Thedissolution rate for Form A in this experiment was low, while the ratesfor Forms B and E were similar to those found in the first experiment.It is believed that in this case, a thin layer of the Form A sample diskmay have converted to Form E upon exposure to water. This is supportedby the evidence of rapid conversion of Form A to Form E in thesolubility experiment. The diffraction pattern of the undissolved solidsdoes not indicate a form change. However, the bulk of the sample disk isnot exposed to water. Therefore, the true intrinsic dissolution rate ofForm A is believed to be close to 0.35 mg per cm² per minute. Aninsufficient quantity of Form A was available to repeat the experiment.

A summary of the intrinsic dissolution rates is given in Table 6. Theamounts dissolved at each time point are summarized in Tables 10 and 11.

TABLE 10 Intrinsic Dissolution Experiment #1 Results Time Point Form A^(a) Form B ^(a) Form E ^(a)  5 min 5.76   10.80 ^(b) 2.70 10 min 7.73 6.85 4.13 20 min 11.31 10.25 6.96 30 min 15.59 14.35 9.60 45 min 21.9820.57 12.57 60 min 27.11 25.70 15.16 90 min 34.17 34.34 20.82 ^(a)Results are reported as Cumulative Amount Dissolved per Unit Area(mg/cm2) ^(b) This date point not included in graph since the value ishigher than the next two data points.

TABLE 11 Intrinsic Dissolution Experiment #2 Results Time Point Form A^(a) Form B ^(a) Form E ^(a)  5 min 4.50 5.04 3.06 10 min 5.22 6.12 4.3120 min 7.54 7.73 11.40 30 min 11.46 12.72 11.93 45 min 15.01 17.33 14.7260 min 18.38 21.93 18.52 90 min 24.38 31.64 26.24 120 min  30.35 41.3133.56 150 min  35.26 49.54 40.82 ^(a) Results are reported as CumulativeAmount Dissolved per Unit Area (mg/cm2)

6.9 Analyses of Mixtures of Polymorphs

This invention encompasses mixtures of different polymorphs. Forexample, an X-ray diffraction analysis of one production sample yieldeda pattern that contained two small peaks seen at approximately 12.6° and25.8° 2θ in addition to those representative of Form B. In order todetermine the composition of that sample, the following steps wereperformed:

-   -   1) Matching of the new production pattern to known forms along        with common pharmaceutical excipients and contaminants;    -   2) Cluster analysis of the additional peaks to identify if any        unknown phase is mixed with the original Form B;    -   3) Harmonic analysis of the additional peaks to identify if any        preferred orientation may be present or if any changes in the        crystal habit may have occurred; and    -   4) Indexing of the unit cells for both Form B and the new        production sample to identify any possible crystallographic        relationships.        Based on these tests, which can be adapted for the analysis of        any mixture of polymorphs, it was determined that the sample        contained a mixture of polymorph forms B and E.

6.10 Dosage Form

Table 12 illustrates a batch formulation and single dosage formulationfor a 25 mg single dose unit of a polymorphic form of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dione.

TABLE 12 Formulation for a 25 mg capsule Percent By Quantity QuantityMaterial Weight (mg/tablet) (kg/batch) Polymorphic Form of 3-(4- 40.0%25 mg 16.80 kg amino-1-oxo-1,3 dihydro- isoindol-2-yl)-piperidine-2,6-dione Pregelatinized Corn Starch, NF 59.5% 37.2 mg 24.99 kg MagnesiumStearate 0.5% 0.31 mg 0.21 kg Total 100.0% 62.5 mg 42.00 kg

The pregelatinized corn starch (SPRESS B-820) and polymorphic form of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)-piperidine-2,6-dionecomponents are passed through a screen (i.e., a 710 μm screen) and thenloaded into a Diffusion Mixer with a baffle insert and blended for about15 minutes. The magnesium stearate is passed through a screen (i.e., a210 μm screen) and added to the Diffusion Mixer. The blend is thenencapsulated in capsules using a Dosator type capsule filling machine.

The entire scope of this invention is not limited by the specificexamples described herein, but is more readily understood with referenceto the appended claims.

1.-28. (canceled)
 29. A composition comprising amorphous3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione andcrystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate.
 30. The composition of claim 29, comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that has an X-ray powder diffraction pattern comprisingpeaks at approximately 16, 22, and 27 degrees 2θ.
 31. The composition ofclaim 30, wherein the crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate has an X-ray powder diffraction pattern further comprising apeak at approximately 18 degrees 2θ.
 32. The composition of claim 29,comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that has an X-ray powder diffraction pattern comprisingpeaks at approximately 15.8, 22.2, and 26.7 degrees 2θ.
 33. Thecomposition of claim 32, wherein the crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate has an X-ray powder diffraction pattern further comprising apeak at approximately 18.2 degrees 2θ.
 34. The composition of claim 29,comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that corresponds to the representative X-ray powderdiffraction pattern provided in FIG.
 6. 35. The composition of claim 29,comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that corresponds to the representative X-ray powderdiffraction pattern provided in FIG.
 32. 36. The composition of claim29, comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that corresponds to the representative X-ray powderdiffraction pattern provided in FIG.
 33. 37. The composition of claim29, comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that corresponds to the representative X-ray powderdiffraction pattern provided in FIG.
 34. 38. The composition of claim29, comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that has a differential scanning calorimetry thermogramcomprising an endotherm with a maximum at about 268° C.
 39. Thecomposition of claim 38, wherein the thermogram further comprises anendotherm corresponding to dehydration.
 40. The composition of claim 29,comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that has a differential scanning calorimetry thermogramcorresponding to the representative differential scanning calorimetrythermogram provided in FIG.
 9. 41. The composition of claim 29,comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that has a differential scanning calorimetry thermogramcorresponding to the representative differential scanning calorimetrythermogram provided in FIG.
 42. 42. The composition of claim 29,comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that has a differential scanning calorimetry thermogramcorresponding to the representative differential scanning calorimetrythermogram provided in FIG.
 43. 43. The composition of claim 29,comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that has a differential scanning calorimetry thermogramcorresponding to the representative differential scanning calorimetrythermogram provided in FIG.
 44. 44. The composition of claim 29,comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that has between approximately 0.46 and approximately 0.59moles of water per mole of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dione.
 45. The composition ofclaim 29, comprising crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate that has a thermal gravimetric analysis curve comprising aweight loss of between about 3.1% and about 4.0% when heated from about30° C. to about 175° C.
 46. The composition of claim 29, wherein thecomposition comprises: a) less than 50% by weight of amorphous3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione; and b)greater than 50% by weight of crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate.
 47. The composition of claim 29, wherein the compositioncomprises: a) less than 25% by weight of amorphous3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione; and b)greater than 75% by weight of crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate.
 48. The composition of claim 29, wherein the compositioncomprises: a) less than 10% by weight of amorphous3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione; and b)greater than 90% by weight of crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate.
 49. The composition of claim 29, wherein the compositioncomprises: a) less than 5% by weight of amorphous3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione; and b)greater than 95% by weight of crystalline3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehemihydrate.